COX-2 expression correlation with invasion in human breast carcinomas and in cell line MDA-MB-231 in vitro

COX-2 expression by means of immunohistochemistry in 120 cases breast invasive ductal carcinomas and 60 cases benign lesions were compared with clinicopathological features and prognostic molecular markers. COX-2 role of invasiveness and chemotaxis in breast cancer cell line MDA-MB-231 by using small RNA interference (siRNA) plasmids to disrupt COX-2 expression in vitro were investigated. The results show that COX-2 immune positivity and percentage of positive cells in breast carcinomas were higher than those in benign lesions and positivity correlated significantly with HIF-1α, VEGF, the grading, metastasis and vascular invasion of carcinoma (p<0.05). This study suggests that COX-2 overexpression correlates with poor clinicopathological parameters in breast cancers, and the reduction of COX-2 expression can obviously inhibit the invasion and chemotaxis of cancer cell line MDA-MB-231 using RNA interference. The findings of the present study suggest that COX-2 overexpression in breast cancer may be considered as a negative prognostic marker.


Introduction
Breast cancer is one of the commonly-encountered malignant tumors (Lakhani et al., 2012;Thorat et al., 2013), and is the second leading cancer causing death in women all over the world (Elston et al., 1991;Yi et al., 2014;Wang et al., 2014).It is mainly a multifactorial disease, occurring as a result of the combined effects of factors (Khan et al., 2015;Aggarwal et al., 2014).During the past decades, there has been an increasing interest in their relationship between the cyclooxygenase type-2 (COX-2) overexpress in tumor and their relationship with the biological behavior (Miglietta et al.,2010;Olivan, et al., 2015).Several studies have suggested that a possible role for COX-2 in carcinogenesis, tumor progression, involved in proliferation, apoptosis, angiogenesis and invasion (Miglietta et al., 2010 ).However, other studies reported no significant correlations between COX-2 expression and histopathological parameters or DFS (Ranger et al., 2004;Kelly et al., 2003).T The role for COX-2 on breast cancer is not fully clear, and studies have shown that the proliferation and metastasis of breast cancer cells is a very complex, multistep process, affected by various factors including genetic and so on (Miglietta et al., 2010;Yu et al., 2014).The aim of this study was to investigate COX-2 expression in human breast cancers tissues and its possible association with clinicopathological features and prognostic molecular markers such as human epidermal growth factor receptor type 2 (HER2/neu), ER, PR, Ki-67 proliferation index, HIF-1 and VEGF, etc., and investigated by reducing COX-2 using RNA interference highly malignant breast cancer cell line MDA-MB-231 transfected with small RNA interference (siRNA) plasmids to disrupt COX-2 expression.

Patients and clinicopathological analyses
One hundred twenty-eight patients diagnosed breast invasive cancer were collected during excision surgery at Rizhao People's Hospital from June 2000 to June 2013.The patients ranged in age from 21 to 78 years, and the mean age was 47.5 ± 5.3 years.The patients' body weight ranged from 42 to 79 kg, and the mean body weight was 55.8 ± 4.7 kg.No patient was treated with radiotherapy, chemotherapy, or endocrine therapy before surgery.Sixty cases of benign lesions were selected as a control group.In the benign breast lesion group, the patient ages ranged from 22 to 74 years, and the mean age was 46.2 ± 5.1 years.The patients' body weight ranged from 42 to 73 kg, and the mean body weight was 55.3 ± 6.7 kg.No significant difference was noted regarding general data such as gender, age, and body weight, indicating good comparability between the observation group and benign breast lesion group (p>0.05).For analyses, we considered the age at diagnosis, menopausal status, tumor size, histological grading, lymph node involvement, stage, status of ER, PR, HER2/neu, HIF-1 and VEGF expression, and the Ki-67 indexin relation to the COX-2 expression.Data concerning ER, PR, HER2/neu, HIF-1 and VEGF status, Ki-67 proliferation index, and histological grade were recorded.Patients assessed for the expression of ER, PR, HER2/neu, survivin, HIF-1 and VEGF were grouped as positive or negative.Regarding the Ki-67 proliferation index, patients were divided into two groups: Those with a Ki-67 proliferation index between 1% and 14% and those with proliferation index >14%.Concerning the histological grade, patients were classified into two groups: GradeⅠor II, Grade III.
The pathological diagnosis was verified by histological methods independently by two pathologists, and pathological categorization was determined according to the current World Health Organization classification system, and the pathological diagnosis was verified by histological methods independently by two pathologists, and the pathologists were blinded to the subject's clinical history and the results of the immunohistochemistry staining assay.The pathological reading was determined for each biopsy slide with an overall pathological diagnosis determined for each subject.The tumor grade was determined according to the modified Bloom-Richardson score (Thorat et al., 2013).The grade was obtained by summing the scores for tubule formation, nuclear pleomorphism, and mitotic count, which were scaled as 1, 2, or 3.The final scores ranged between 3 and 9 and were then divided into three grades (I-III).The final grading scores were as follows: Sum of points, 3-5, final grade I; 6-7, II; and 8-9, III.The pathologists were blinded to the subject's clinical history and the results of the immunohistochemistry staining assay.According to the International Union against Cancer (UICC) TNM classification of solid malignant tumors standard (7th ed) and American Joint Committee on Cancer (AJCC) Cancer Staging Manual (7th ed) were used to evaluate the therapeutic effect.

Immunohistochemistry
Breast tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin at 4°C for 24 hours.Tissue sections at 5 μm thickness were deparaffinized and rehydrated using standard procedures.The specimens were examined under a binocular-dissecting microscope.Immunoreactions were processed using the Ultra Sensitive TM S-P Kit (Maixin-Bio, China) according to the manufacturer's instructions to detect differences in tumor tissue COX-2, HIF-1 , VEGF and CD105 expression, and signals were visualized using the DAB substrate, which stains the target protein yellow.The pathological specimens were reviewed independently by two pathologists and the pathologists were blinded to the subject's clinical history, and the results of the immunohistochemistry staining assay.
For COX-2, the immunoreactive score (IRS) was obtained by multiplying the percentage of positive cells and the staining intensity.Immunostaining was recorded according to stain intensity (intensity score) and percentage of cancerous cells that stained positively (quantity score).Briefly, a proportion score was assigned that represented the estimated proportion of positive tumor cells on the entire slide.For each histological section, the percentage of positive cells was scored as 0 (<5%), 1 (6%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%), and the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).Immunohistochemical results with an IRS of 0 were considered negative, an IRS of 1-4 was considered weakly positive, an IRS of 5-8 was considered moderately positive, and an IRS of 9-12 was considered strongly positive.The negative controls were used.The primary antibody was replaced with PBS, containing 0.1% bovine serum albumin at the same concentration as the primary antibody.The positive controls were tissues known to express the antigen being studied.
The immunoreactivity of positivity for VEGF was indicated by cytoplasmic staining, and HIF-1 was expressed as the percentage of cancer cells showing nuclear reactivity.For VEGF and HIF-1 , cells were classified according to the positive rate and color intensity as follows: Negative, number of positive cells ＜25%; positive, brown particles, number of positive cells ≥25%.
For ER, PR, and Ki-67 expression, the percentage of cancer cells showing nuclear reactivity was recorded after inspection of all of optical fields at ×200, and the mean value was used to score each case.Tumors showing expression in >1% of cancer cells were considered positive.ER and/or PR positivity was considered combined ER and PR positivity, and combined ER and PR negativity was considered both ER and PR negativity.For the Ki-67 proliferation index, patients were divided into two groups: Those with a value between 1% and 14% and those with a value >14%.
Histologically recognizable vessels within tissue sections served as internal controls for CD105 immunostaining.The MVD was evaluated under light microscopy according to the procedure described (Kopczyńska and Makarewicz, 2012).Briefly, after scanning the sections at low magnifications, five tumor areas with the greatest number of distinctly highlighted micovessels were selected.The number of vessels was counted in the highlighted micovessels at high magnifications (400×), and the average counts of the fields were recorded.Each brown-stained endothelial cell or endothelial cell cluster, which was clearly separate from the adjacent micovessels, tumor cells and connective tissue elements was considered a single, countable microvessel.Areas of fibrosis, necrosis and inflammation, and vessels with muscular walls were not counted.
In addition, regarding the cell membrane reactivity of HER2/neu, oncoprotein expression was evaluated following a similar approach, and the mean value was used to score each case.Tumors expressing HER2/neu in >10% of the cancer cells were considered to show positive expression.

In vitro studies by reducing COX-2 by using RNA interference in cells line MDA-MB-231
In vitro studies, to investigate the role of COX-2 in invasiveness and chemotaxis of the highly malignant breast cancer cell line MDA-MB-231 RNA interference was used.The MDA-MB-231 cells were cultured.The small interfering RNA expression vectors targeting COX-2 was constructed.MDA-MB-231 cells were transfected with small RNA interference plasmids to disrupt COX-2 expression.COX-2 mRNA levels were detected using RT-PCR.Chemotaxis assay and scratch assay were examined to detect the migration and chemotaxis ability of MDA-MB-231 cells.The adhering ability of MDA-MB-231cells was examined using adhesion assay.The in vitro invasion ability of MDA-MB-231 cells was examined using matrigel invasion assay.

Statistical analysis
SPSS version 17.0 statistical software (SPSS Inc., USA) was used to analyze the data.Enumeration data with chi-squared (χ 2 ) test.The relationship of this dichotomous variable to other clinicopathological correlates was established using χ 2 or Fisher's exact tests, as appropriate.The MVD results were expressed as the mean and standard deviation (SD).Because the results which were measured in tumor tissues, was not Gaussian, the nonparametric Mann-Whitney U-test was used to determine differences between the benign and malignant groups.Kaplan-Meier overall survival curves were constructed to demonstrate the survival differences between the COX-2 positive and COX-2 negative patients.All other statistical tests were performed using Graphpad Prism 5.0 (Graphpad Software Inc., USA).A p value less than 0.05 was deemed statistically significant.

Clinical features and follow-up date
A summary of the results concerning major clinicopathological parameters, including pathological tumor stage, nodal status, hormone receptor status, tumor size, grading and vascular space involvement is provided in Table I.

COX-2 Immunohistochemical express and relationship with clinicopathological factors COX-2 express in different groups
COX-2 immune positivity in tumor cells was appreciated by immunohistochemistry as a diffuse, granular cytoplasmic staining in breast lobules.The expressions of COX-2 in observation and control groups were shown in Figure 1.Benign tissues, COX-2 was weekly expressed only 15.4% (10/60).Immunohistochemical analysis revealed some degree of positivity in 78.9% (101/128) of the tumors examined, and the positive rates of COX-2 in breast carcinomas were higher than in benign tissues (p<0.05).The degree of COX-2 expression differed between Grade III and Grade I or II, the normal tissue away from the tumor did not stain with COX-2 (results not shown).

Correlations between COX-2 positivity and clinicopathological factors in breast cancers
Relationship between COX-2 and clinicopathological parameters of breast cancer was shown in Table II.Statistical analysis revealed significant relationships between COX-2 expression and clinical parameters such as histoligical grading, extent of primary tumor, vascular invasion, lymph node metastasis, distant metastasis  (p>0.05).

Correlation between COX-2 and molecular markers
The correlation between COX-2 and molecular markers expresses in breast carcinoma was shown in Table II.COX-2 immune positivity correlated significantly with HIF-1 , VEGF, the grading, lymph node metastasis, distant metastasis and vascular invasion of carcinoma (p<0.05,respectively), but not with biological parameters such as age, size of tumor, ER, PR and HER 2 (p>0.05).

COX-2 and MVD assessment in breast cancer patients
The tissues of breast cancer and benign leisions were immunohistochemically stained for CD105 and MVD was assessed based on the number of CD105-positive vessels.CD105 positivity was indicated by membrane staining.Immunohistochemical results in observation and control groups were listed and shown in Figure 2A.
The MVD had statistical significance in invasive breast cancer (31.7 ± 8.6) and benign breast leisions (10.0 ± 4.0) groups (t =22.17, p<0.01).MVD in the peripheral area adjacent to the lesion was significantly higher than those central area within the lesion in every group (p<0.01),shown in Figure 2B.MVD in COX-2 positive was significantly higher than those in negative in breast cancer patients shown in Figure 2C.

Survival analysis
Patients were monitored for survival from 2000 to 2014 through telephone communication and periodic returns to the rizhao people's hospital.or death from any cause.Because of the possibility that some patients with no evidence of disease may have artificially influenced the PFS, we checked for the heterogeneity in PFS among patients grouped by their treatment before entering the study (no local treatment vs. surgical or local radiotherapy vs. whole-breast radiotherapy).Among 86 evaluable patients 58 patients achieved SD, 28 patients achieved PD, and 55 patients experienced some degree of radiologic improvement.In all patients, the overall response rate was 37.2%, with a median PFS of 23 months (95% CI = 11.3-26.0)and a median OS of 62 months (95% CI = 42.7-67.2).For COX-2 expression, the patients were divided into negative and positive two groups.Considering only the COX-2 expression, survival was found to be longer in negative group than that in positive group, and there were slightly statistically significant between 95% CI = 1.715-2.843,the two groups (χ 2 = 9.567, p = 0.0448).Kaplan-Meier survival curves demonstrated survival differences between COX-2 positive and COX-2 negative patients (p = 0.0448), shown in Figure 3.Other factors which may be associated with COX-2 in breast cancer, which should be investigated in further studies.

In vitro studies by reducing COX-2 by using RNA interference in cells line MDA-MB-231
The results of restriction endonuclease digestion electrophoresis and DNA sequencing showed that the interferenced plasmid siCOX-2 was constructed successfully.The cell strains transfected were named as MDA-MB-231/basic and MDA-MB-231/siCOX-2 (Figure 4AB).The levels of COX-2 mRNA detected by RT-PCR shown in Figure 4C.The results of RT-PCR showed that the levels of COX-2 mRNA of MDA-MB-231/siCOX-2 (0.9 ± 0.1) were obviously reduced at 48 hours after transfection, compared to MDA-MB-231/basic cells (0.3 ± 0.1) (p<0.01).
When a scratch was created in the fluent monolayer cells, it took the COX-2-reduced MDA-MB-231 cells a longer time to fill the gap in the scratch assay (p<0.05).Figure 5A showed COX-2-reduced MDA-MB-231 cells showed decreased chemotaxis ability compared with the control cells (p<0.01).The COX-2-reduced MDA-MB -231 cells which were induced by EGF showed decreased chemotaxis and directional migration ability by using RNA interference.Adhesion assay showed in Figure 5B.The number of the adhering cells was decreased for reduction of COX-2 within 5 and 15 minutes (p<0.01).The adhering ability of the COX-2reduced MDA-MB-231 cells was reduced obviously when the cells were adhered in fibronectin.Using RNA interference, the reduction of COX-2 expression can obviously inhibit the invasion and chemotaxis of the highly malignant breast cancer cell line MDA-MB-231 in vitro (Figure 5C).The invasion assay showed prominent differences between the COX-2-reduced MDA-MB-231 cells and the control cells (p<0.01).

Discussion
The pathogenesis of breast cancer is not fully clear, studies have shown that the proliferation and metastasis of breast cancer cells is a very complex, multistep process, affected by various factors including the common role of environmental, genetic and so on (Sueoka et al., 2013).It is critical for breast cancer management to identify those biomarkers that would provide, at the time of diagnosis, a reliable prognostic assessment and prediction of treatment response (Diers et al., 2013;Cao et al., 2014;Witteveen et al., 2015).
Several studies have shown that COX-2 is implicated in carcinogenesis and tumor progression in many human cancers, including breast cancer.However, other studies report there is no significant correlations between COX-2 expression and histopathological parameters or disease-free survival, moreover, roles for COX-2 in invasion and with other molecular biomarkers remain unclear.In the current study, the results show that COX-2 overexpression in breast cancers correlates with poor clinicopathological parameters such as invasion and metastasis in vivo and also obviously inhibit the invasion and chemotaxis by reduction of COX-2 expression of cancer cell line MDA-MB-231 using RNA interference in vitro.In addition, This study data demonstrated that the positivity correlated significantly with HIF-1 , VEGF in breast cancer.Therefor, COX-2 overexpression in breast cancer may be considered as a negative prognostic marker.
In the current report, tentatively put forward COX-2 overexpression role on invasivenes, the study was disigned to investigate COX-2 overexpression on invasion and metastasis in human breast cancers tissue, and further invasivenes the influences of disruption of COX-2 express using siRNA plasmids to disrupt COX-2 expression to show RNA interference on invasiveness of highly malignant breast cancer cell line in vitro.In human breast tissue study, its possible COX-2 association with clinicopathological features and prognostic molecular markers such as ER, PR, HER2, HIF-1 , VEGF and MVD was also performed.This results indicate that in human tissues, the COX-2 immune positivity and percentage of positive cells in breast invasive ductal carcinomas was 78.9% with moderate positive to strong some degree of positivity, in contrast, in benign lesions tissues, COX-2 was weekly expressed only in 16.7%, the expression of COX-2 in cancer tissues was obviously higher than the expression in benign lesions tissues, the difference was statistically signifi- cant.These data which can be consistent with the notion that a possible role for COX-2 in carcinogenesis.
In this study, next, the effect of COX-2 expression in breast cancer with clinicopathological features, including histological grade, clinical, lymph node metastasis, distant metastasis and vascular invasion etc., difirrent biological behavior were examined.Results indicate that COX-2 expression immune positivity correlated significantly with HIF-1 , VEGF, the grading, lymph node metastasis, distant metastasis and vascular invasion in breast carcinoma, and shorter overall survival in carcinomas patients group related to COX-2 overexpression in tumors.The finding was consistent with the previous finding that demonstrate that COX-2 overexpression in breast cancers correlates with poor clinicopathological parameters such as invasion and metastasis.On the other hand, to explore the function of reducing COX-2 expression can possible obviously inhibit the invasion, next, set out to performed experiment using siRNA plasmids to disrupt COX-2 expression in vitro role studies.RT-PCR analysis showed COX-2 mRNA levels of siCOX-2 were obviously reduced at 48 hours after transfection; the COX-2reduced MDA-MB-231 cells showed decreased chemotaxis ability (p<0.01); the invasion assay showed prominent differences between the COX-2 reduced MDA-MB-231 cells and the control cells (p<0.01).Thus, at current time, the finding was consistent with the previous finding that COX-2 overexpression in breast cancers correlates with poor clinicopathological parameters such as invasion and metastasis; The data demonstrated that the reduction of COX-2 expression can obviously inhibit the invasion and chemotaxis of cancer cell line MDA-MB-231.Taken together, these data indicate that COX-2 overexpression in breast cancer may be considered as a negative prognostic marker.COX-2 is an inducible enzyme that interferes with tumor development and angiogenesis, related to the inhibition of apoptosis through inhibition of the proapoptotic Bax protein and overexpression of the antiapoptotic bcl-2 protein.COX-2 catalyzes the conversion of arachidonic acid to PGE2 and enhances the metastatic phenotype of both breast cancer cells in vitro and breast tumors (Mitchell et al., 2010;Miglietta et al., 2010).PGE2, the catalytic product of COX-2, may promote tumor development and angiogenesis (Boland et al., 2004;Miglietta et al., 2010;Yu et al., 2014).It has been investigated in several human cancers and also correlated with the evolution of the disease (Boland et al., 2004).
Some authors reported that COX-2 up-regulation in a wide variety of malignant solid tumors, including colon, gastric, breast cancer, and its overexpress leads to increased production of prostaglandins that are involved in different physiological and pathophysiological processes, suggesting a possible role for COX-2 in proliferation, carcinogenesis, development, apoptosis, angiogenesis, invasion, and metastasis.In the findings of the present study, we confirm that human breast carcinomas aberrantly express COX-2, and that raised tissue levels of COX-2 may have prognostic value.Patients expressing high levels of COX-2 can develop local recurrence, and have reduced diseasefree and disease-related overall survival.Breast cancer is a heterogeneous disease with multiple genetic alterations able to affect tumor growth, progression and metastasis.However, the pathogenesis of breast cancer is not fully clear, studies have shown that the proliferation and metastasis of breast cancer cells is a very complex, multistep process, affected by various factors including the common role of environmental, genetic and so on (Miglietta et al., 2010).The findings of the present study suggest that the increased expression of COX-2 and HIF-1 , VEGF and MVD-CD105 positively expressive correlation played an important role in the development of breast cancer and might be responsible for the enhanced activity in tumor-induced neovascularization, invasion and metastasis.
We tentatively put forward that increased COX-2, HIF-1 , VEGF and MVD may serve as valuable indicators of biological behavior of breast cancer.Hypoxia, is a common feature of various cancers.Solid tumors are characterized by regions of low oxygen tension, which play a central role in tumor progression and resistance to therapy.Cells under hypoxic conditions develop numerous adaptive responses to hypoxic stress concurrently with altered expression of hundreds of genes that are regulated by hypoxia inducible factors (Sueoka et al., 2013;Majmundar et al., 2010).Low oxygen tension affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low oxygen tension to maintain the cellular bioenergetics.HIF-1 is an important cellular survival protein under hypoxic conditions, regulating the cellular response to low oxygen tension via recruitment of a transcriptional co-activator, induces expression of multiple genes involved in cell survival, proliferation, angiogenesis, and tumor development (Dewi and Fatchiyah, 2013;Semenza et al., 2010).Angiogenesis is essential for the growth and metastasis of major solid tumors and has been correlated with prognosis in human cancer (Leo et al., 2006) (Kelly et al., 2003).Due to an intimate association with genesis, development, metastasis and infiltration, it is an important indicator to judge the cancer metastasis and infiltration in clinic (Thielemann et al., 2013;Wang et al., 2013;Wang et al., 2014).In the study, VEGF expression in breast carcinoma (60.9%) was higher than that in benign tissue and the expression of VEGF was positively correlated with COX-2 in breast carcinoma (p<0.05).In our study, we also investigated the prognostic significance of CD105 and MVD assessed based on the number of CD105-positive vessel in tissue of breast cancer and epithelium adjacent to the lesion.The MVD in COX-2 positive group was higher than that in COX-2 negitive group, there were slightly correlated with MVD.Tumor angiogenesis and its clinical significance have been studied in a variety of neoplasms.Angiogenesis contributes to the pathogenesis of various cancer, and microvessel density may improve our ability to predict breast cancer extension.Microvessel densities are significantly greater in the primary tumors of patients with metastatic disease than in those without metastases.This conformed that the overexpression COX-2 in breast cancer might be important biological markers for invasion and lymph node metastasis, and the combined detection of COX-2, HIF-1 , VEGF and MVD are benefit to better prewarning markers for monitoring their metastasis in clinic judging the patients with breast carcinoma (Koyama et al., 2010;Dallas et al., 2008;Fonsatti et al., 2010).
Breast cancer is mainly a multifactorial disease, occurring as a result of the combined effects of factors.Many studies have found that certain biological indicators by detecting abnormal expression of HER2/ neu, ER, PR etc., can guide clinical diagnosis and treatment activities prognosis.In addition, several reports have described that in ER -negative patients, but not in ER-positive ones, up-regulation of COX-2 is associated with reduced DFS (Yu et al., 2014).In ERnegative tumors, increased COX-2 expression may contribute to breast cancer progression and the suggested mechanisms include increased prostaglandin production, which, in turn, would promote angiogenesis, and ultimately metastasis (Kim et al., 2014).However, our results in this study suggest that COX-2 immune positivity not significantly correlated with HER2/neu, ER, PR.Moreover, several studies have shown that increased levels of COX-2 are frequently associated with younger patient age, generally, with a more aggressive phenotype (Costa et al., 2002).
Unfortunately, our results suggest that COX-2 immune positivity not significantly correlated with biological parameters such as age, size of tumor, etc.We have to point out that we do not detect every aspect of test cases, and we only detect few biological parameters in the present study.It also should be noted that the number of cases is too limited, and this study has examined only 128 cases infiltrating ductal carcinoma, not included the subtype of breast carcinomas.Other factors which may be associated with COX-2 in breast cancer, which should be investigated in further studies.
It is critical for breast cancer management to identify those biomarkers that would provide those factors potentially curable given the availability of early diagnosis and effective therapeutic protocols, at the time of diagnosis, a reliable prognostic assessment and prediction of treatment response (Thorat et al., 2013;Peng et al., 2013 ).
In this study, the results revealed that patients with increased COX-2 expression have shorter survival times.A slightly correlation between COX-2 expression and prognostic factors in breast cancer suggesting that increased COX-2 expression was related to worse prognosis.The result suggests that the difference between COX-2 expression in the breast cancer may related to the differentiated behavior of these tumors, confirming the association between the COX-2 expression and disease aggressiveness.This study showed shorter survival in patients whose tumors expressed more COX-2.Therefore, there are possibility of using COX-2 inhibitor anti-inflammatory drugs to treat mammary tumors.When overall survival was assessed, the results suggesting that COX-2 is essential for the development and evolution of neoplastic disease, resulting in shorter overall survival and worse prognosis.This study suggests that the increased expression of COX-2 in tumors indicates a higher risk of development and dissemination of the disease.

Conclusion
COX-2 overexpression correlates with poor clinicopathological parameters such as invasion and metastasis in breast cancers, and the reduction of COX-2 expression can obviously inhibit the invasion and chemotaxis of cancer cell line MDA-MB-231 using RNA interference.The findings of the present study suggest that COX-2 overexpression in breast cancer may be considered as a negative prognostic marker.

Ethical Issue
The study protocol was endorsed by the Medical Ethical Committee of the Hospital; besides, each participant provided informed consent.

Figure 2 :Figure 3 :
Figure 2: The MVD comparison in different groups.p<0.01 comparison between benign and cancer (A), comparison between central and peripheral area in cancer (B), and comparison between COX-2 positive and COX-2 negative in breast cancer Figure 4: The constructed interferenced plasmid siCOX-2 and levels of COX-2 mRNA.Results of identification of COX-2 with Bgl II and Hind III (A); The detection of cell's totall RNA (B); RT-PCR analysis showed COX-2 mRNA levels of siCOX-2 were obviously reduced at 48 hours after transfection (C)

Figure 5 :
Figure 5: Chemotaxis, adhesion and invasion ability of COX-2 reduced MDA-MB-231 cells by siRNA.COX-2-reduced MDA-MB-231 cells showed decreased chemotaxis ability compared with the control cells (A).Adhesion assay showed the number of the adhering cells was decreased for reduction of COX-2 within 5 and 15 min (B).Comparison of disruption of COX-2 by the siRNA impaired invasion into matrivgel in the MDA-MB-231/basic and MDA-MB-231/ siCOX-2 cells (C)

Table I Clinicopathological data of patients
Figure 1: COX-2 immunohistochemical staining in tissue sections of breast carcinoma